Quiz 4

Question 1

Describe agrose gels

Answer 1

Repeating units of D-galactose and 3,6-anhydrogalactose
Forms a microporous matrix which allows rapid diffusion of high molecular weight macromolecules without significant respriction by the gel
Concentration of Agrose determines average pore size

Question 2

Describe gel red

Answer 2

Safer than ethidium bromide
Does not penetrate cells
More sensitive and stable

Question 3

Describe nanodrop

Answer 3

Small spectrophotométer

Can measure concentration and purity of protein and nucleic acids with UV absorbance

Question 4

Describe plasmid purification’s

Answer 4

DNA h-bonds with matrix in column under high salt concentration
DNA h-bonds with water and comes off matrix under low salt conditions
DNA absorbance ratio of 260nm to 280nm should be around 1.8 (accepted as pure)

Question 5

What are the advantages and disadvantages of mammalian expression of plasmid vectors?

Answer 5

Ad: post-translational modifications present in mammalian cells, can localize fusion proteins
Dis: produces much smaller amounts of proteins than general bacteria

Question 6

Describe mammalian expression

Answer 6

Strong mammalian promoter tend to be viral promoters
Multiple cloning sites
Poly-A tail
Antibiotic resistance
Origin of replication in bacterial cells
Amino acid tags to be added at either n-terminus or c-terminus
Fusion protein

Question 7

What are the advantages and disadvantages of bacterial expression?

Answer 7

Ad: produces much larger amounts of protein, easy to transform
Dis: does not have post translational modifications, can not be used to localize fusion proteins

Question 8

Describe bacterial expression

Answer 8

Strong bacterial promoters
Multiple cloning sites
Antibiotic resistance
Origin of replication
Amino acid tags added to either terminus
Fusion protein
B-galactosidase gêne for transformation efficiency

Question 9

What are some reporters?

Answer 9

Luciferase
B-galactosidase
B-glucuronidase
Green fluorescent protein

Question 10

Describe RNA interference

Answer 10

Short hairpin loop rna interferes constructs
Target sequence sense
Target sequence antisense

Question 11

Describe dihydrofolate reductase

Answer 11

Converts dihydrofolate into tetrahydrofolate by the addition of a hydride from NADPH
THF is a methyl group shuttle required for synthesis of nucleotides and amino acids

Question 12

How is DHFR associated with cancer?

Answer 12

DHFR inhibition or reduction disrupts nucleic acid synthesis affecting cell growth and proliferation
Methotrexate (one of the first chemotherapeutic agents)- inhibits DHFR

Question 13

What regulates the DHFR system?

Answer 13

LacI

Question 14

Describe the chromatography basics

Answer 14

Mobile phase (solvent and the molecules to be separated)
Stationary phase (through which the mobile phase travels)
Molecules travel through the stationary phase at different rates because of their chemistry

Question 15

Name some different types of protein purification

Answer 15

HPLC: amino acids by charge, size or hydrophobicity,
Ion exchange: proteins by charge, change salt concentration or pH
Gel filtration: protein by molecular weight
Affinity: proteins by affinity for ligand, excess free ligand

Question 16

What are the steps to affinity purification?

Answer 16

Pour column
Wash resin to remove packing buffer
Équilibriste resin
Bind GST-DHFR-His
Elute unbound proteins
Wash protein bound onto the resin
Elute GST-DHFR-His

Question 17

What is beers law?

Answer 17

A= ECI
E- molar absorbtivity
I- path length
C- concentration of compound in solution

Question 18

Describe simple optical density measurement

Answer 18

Absorbance at 280nm or 205nm provides a rough approximation of protein concentration